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The non-flavonoid inducible nodA3 and the flavonoid regulated nodA1 genes of Rhizobium tropici CIAT 899 guarantee nod factor production and nodulation of different host legumes

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Abstract

Aims

The symbiosis between rhizobia and their host legumes is initiated by a complex molecular dialogue in which the activation of bacterial NodD proteins by appropriate plant flavonoids triggers the expression of the bacterial nodulation (nod) genes. These genes are involved in the synthesis and export of the Nodulation factors (NF), which are signal molecules that, when recognized by plant receptors, launch the symbiotic process. The core of NF is synthesized by proteins encoded by the nodABC genes, whereas the rest of the nod genes protein products are involved in the decoration of NF with different chemical substituents and their export to the environment. Rhizobium tropici CIAT 899, the microsymbiont of common bean (Phaseolus vulgaris), is characterized for tolerating multiple physical stresses and for synthesizing a large variety of NF not only in the presence of inducing flavonoids but also when high concentrations of salt are present. In addition, another interesting feature of the R. tropici CIAT 899 genome is the presence of three different nodA genes on the symbiotic plasmid, although their exact roles remain to be elucidated. In this work, we characterize the role of the three NodA proteins of R. tropici CIAT 899 in symbiosis.

Methods

We have analyzed by quantitative PCR the expression of the nodA1, nodA2 and nodA3 genes of CIAT 899, showing that only nodA1 and nodA2 are inducible by flavonoids. We have also constructed single, double and triple non-polar mutants in these genes in order to study their importance in NF production and in nodulation of four different host legumes of CIAT 899: P. vulgaris, Leucaena leucocephala, Lotus japonicus, and L. burttii.

Results

The nodA1 and nodA2 genes of CIAT 899 (both inducible by flavonoids) are more related between them than with nodA3 (non-flavonoid inducible). Interestingly, the presence of NodA1 or NodA3 is crucial for nodulation with L. leucocephala and L. japonicus, since both NodA1 or NodA3 guarantee Nod factor production upon apigenin induction. Interestingly, osmotic-stressing conditions increase the quantity and diversity of the Nod factors synthesized by NodA2, being these molecules able to induce the formation of nodule primordia on P. vulgaris.

Conclusions

We concluded that R. tropici CIAT 899 requires at least the flavonoid-induced nodA1 or the flavonoid-independent nodA3 genes for ensuring symbiotic success in the four assayed host-legumes. The inducible nodA2 gene is sufficient to induce nodulation on P. vulgaris and L. burttii but not in L. leucocephala and L. japonicus. To our knowledge, in this work we report for the first time that a non-flavonoid inducible copy of nodA is enough to induce nodulation on legumes.

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Acknowledgments

This work was supported by the Ministerio de Economía y Competitividad of the Spanish government (projects AGL2016-77163-R and BIO2016-78409-R). P. del Cerro is recipient of a FPU fellowship (FPU14/00160) of the Ministerio de Economía y Competitividad. Authors sincerely thank Servicio General de Biología of the CITIUS from the University of Seville for allowing us to use their laboratory equipment.

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Authors and Affiliations

  1. Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, Apdo. Postal 4012, Sevilla, Spain

    Pablo del Cerro, Paula Ayala-García, Irene Jiménez-Guerrero, Francisco Javier López-Baena, José María Vinardell, Manuel Megías, Francisco Pérez-Montaño & Francisco Javier Ollero

  2. Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, 76100, Rehovot, Israel

    Irene Jiménez-Guerrero

  3. Embrapa Soja, 86001-970, Londrina, Paraná, CP, 231, Brazil

    Mariangela Hungria

  4. Departamento de Química Orgánica. Facultad de Química, Universidad de Sevilla, C/ Profesor García González, 1, Apdo. Postal, 41012, Sevilla, Spain

    Antonio M. Gil-Serrano

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  1. Pablo del Cerro
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Correspondence to Francisco Javier Ollero.

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Supplementary file 1

Bacterial strains and plasmids used in this study. (DOCX 16 kb)

Supplementary file 2

Primers used in this study. (DOCX 17 kb)

Supplementary file 3

Confirmation of deletion events in the nodA mutants by PCR and hybridization. A. PCR amplification by using nodA gene external primers (primers -A and -D listed in Supplementary file 2) and gDNA of the different mutant backgrounds as templates. Amplicons were confirmed by sequencing. B. Southern blot hybridization with probes of nodA genes (made with primers -A and -D listed in Supplementary file 2). gDNA of each nodA mutant strain was digested with specific restriction enzymes. (PNG 2994 kb)

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Supplementary file 4

Venn diagram showing the overlapping of NF synthetized in the presence of apigenin by the different CIAT 899 double nodA mutants. (XLSX 488 kb)

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del Cerro, P., Ayala-García, P., Jiménez-Guerrero, I. et al. The non-flavonoid inducible nodA3 and the flavonoid regulated nodA1 genes of Rhizobium tropici CIAT 899 guarantee nod factor production and nodulation of different host legumes. Plant Soil 440, 185–200 (2019). https://doi.org/10.1007/s11104-019-04073-2

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